Cysteine or thiol proteases contain a cysteine residue at the active site responsible for proteolysis. Since cysteine proteases have been implicated in a number of diseases, including arthritis, muscular dystrophy, inflammation, tumor invasion, glomerulonephritis, malaria, and other parasite-borne infections, methods for selectively and irreversibly inactivating them provide opportunities for new drug candidates. See, for example, Meijers, M. H. M. et al., Agents Actions (1993), 39 (Special Conference Issue), C219; Machleidt, W. et al, Fibrinolysis (1992), 6 Suppl. 4, 125; Sloane, B. F. et al., Biomed. Biochim. Acta (1991), 50, 549; Duffy, M. J., Clin. Exp. Metastasis (1992), 10, 145; Rosenthal, P. J., Wollish, W. S., Palmer, J. T., Rasnick, D., J. Clin. Investigations (1991), 88, 1467; Baricos, W. H. et al, Arch. Biochem. Biophys. (1991), 288, 468; Thornberry, N. A. et al., Nature (1992), 356, 768.
Low molecular weight inhibitors of cysteine proteases have been described by Rich, Proteinase Inhibitors (Chapter 4, "Inhibitors of Cysteine Proteinases"), Elsevier Science Publishers (1986). Such inhibitors include peptide aldehydes, which form hemithioacetals with the cysteine of the protease active site. The disadvantage of aldehydes is their in vivo and chemical instabilities.
Aldehydes have been transformed into .alpha.,.beta.-unsaturated esters and sulfones by means of the Wadsworth-Emmons-Horner modification of the Wittig reaction (Wadsworth, W. S. and Emmons, W. D. (J. Am. Chem. Soc. (1961), 83, 1733: Equation 1). ##STR1##
where
R=alkyl, aryl, etc. PA1 EWG=COOEt, SO.sub.2 Me, etc. PA1 PG=protecting group PA1 R.sub.1, R.sub.2 =amino acid side chains PA1 X=Cl, F, N.sub.2, OC(O)R, (+)S(CH.sub.3).sub.3 PA1 R.sub.10 is hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid residue with or without an amino end blocking group or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 A--B is a peptide linkage; and PA1 EWG is an electron withdrawing group. PA1 R.sub.10 is hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid residue with or without an amino end blocking group or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 R.sub.1 is an ester moiety; and PA1 the A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.10 is hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X an R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 R.sub.2 is a side chain moiety; and PA1 the A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.10 is hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; and PA1 the A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.10 hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 R.sub.3 is an ester moiety; and PA1 the A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.10 =hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X, R.sub.11 and Z are amino acid side chains, with either (R) or (S) stereochemical configuration, PA1 Q=hydrogen, an ester, a peptide residue, or an amide moiety; and PA1 The A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.10 =hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 R.sub.4 =ketone moiety; and PA1 the A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.10 =hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 the C.ident.N group is a nitrile; and PA1 the A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.10 =hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 R.sub.5 =sulfoxide moiety; and PA1 the A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.10 =hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X, R.sub.11 and Z are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 Q=hydrogen, an ester, a peptide residue or an amide moiety; and PA1 the A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.10 =hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 R.sub.6 and R.sub.7 a re sulfoximine moieties; and PA1 the A--B linkage is a peptide residue or an isosteric form thereof. PA1 R.sub.8 =the five or six membered homo- or heterocyclic aromatic rings with at least one substituted EWM, MDG, or DG; and PA1 R.sub.9 =a suitable group as defined below. PA1 R.sub.10 =hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 the A--B linkage is a peptide residue or an isosteric form thereof; PA1 D=an oxygen, sulfur, nitrogen, phosphorus or arsenic atom; and PA1 EWM=an electron withdrawing moiety. PA1 R.sub.10 =hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 the A--B linkage is a peptide residue or an isosteric form thereof; PA1 EWM=an electron withdrawing moiety; PA1 MDG=a meta directing group; and PA1 DG=a deactivating group. PA1 R.sub.10 =hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 the A--B linkage is a peptide residue or an isosteric form thereof; PA1 T=a nitrogen or phosphorus atom; and PA1 EWM=an electron withdrawing moiety. PA1 R.sub.10 is hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 A--B is a peptide linkage; and PA1 EWG is an electron withdrawing group. PA1 R.sub.10 is hydrogen, a peptide amino end blocking group, a peptide residue with or without an amino end blocking group, a single amino acid with or without an amino end blocking group, or a label; PA1 X and R.sub.11 are amino acid side chains, with either (R) or (S) stereochemical configuration; PA1 A--B is a peptide linkage; and PA1 EWG is an electron withdrawing group. PA1 where K.sub.I is a dissociation constant with units of M. PA1 k.sub.irr is the first order rate constant of inactivation of E. When [I]&lt;&lt;K.sub.I, the rate equation reduces to ##EQU6## PA1 R=an amino acid side chain: alkyl, aryl, aminoalkyl, etc.; PA1 a) Cl--H.sub.2 N+(Me)OMe, dicyclohexylcarbodiimide, triethylamine; and PA1 b) lithium aluminum hydride. PA1 R.sub.12 is hydrogen or a peptide amino end blocking group; PA1 R.sub.11 any amino acid side chain except glycine (hydrogen), or, when the EWG is a vinylogous ester, phenylalanine (benzyl); and PA1 EWG is an electron withdrawing group. PA1 a) Cl--NH.sub.2 +(CH.sub.3)OCH.sub.3, dicyclohexylcarboiimide, Et.sub.3 N/CH.sub.2 Cl.sub.2 ; PA1 b) LiAlH.sub.4 /THF; PA1 c) NaH/THF; PA1 d) HCl/dioxane/CH.sub.2 Cl.sub.2 ; and PA1 e) 4-methylmorpholine, isobutyl chloroformate/THF. PA1 a) NaH/THF; PA1 b) HCl/dioxane; and PA1 c) Mu-PheOH, 4-methylmorpholine, isobutyl chloroformate/THF. PA1 a) (CH.sub.3).sub.3 SiCH.sub.2 CH.sub.2 OH, dicyclohexylcarbodiimide (DCC), (C.sub.2 H.sub.5).sub.3 N, 4-dimethylamino-pyridine, CH.sub.2 Cl.sub.2 ; PA1 b) HCl/dioxane; PA1 c) NaOH/C.sub.2 H.sub.5 OH; PA1 d) PheOSET, DCC, (C.sub.2 H.sub.5).sub.3 N, CH.sub.2 Cl.sub.2 ; PA1 e) NaH, EPAc-PheOSET, THF; PA1 f) Mu-PheOH, 4-methylmorpholine, isobutyl chloroformate, THF; and PA1 g) (n-C.sub.4 H.sub.9).sub.4 N+F-, 3 .ANG. molecular sieves, THF. PA1 a) Cl--H.sub.2 N+(Me)OMe, dicyclohexylcarboniimide, triethylamine; and PA1 b) lithium aluminum hydride. PA1 a) heat at reflux; PA1 b) solvent; PA1 c) H.sub.2 O/NaOH; PA1 d) organic extraction; PA1 e) mix; PA1 f) HCl/dioxane 4M; PA1 g) couple; PA1 h) base; and PA1 R.sub.8 =the five or six homo- or heterocyclic aromatic rings with at least one substituted EWM, MDG, or DG; and PA1 R.sub.9 =a suitable group as defined above.
.alpha.,.beta.-unsaturated esters (Hanzlik et al., J. Med. Chem., 27(6):711-712 (1984), Thompson et al., J. Med. Chem. 29:104-111 (1986), Liu et al., J. Med. Chem., 35(6):1067 (1992)) and an .alpha.,.beta.-unsaturated sulfones (Thompson et al., supra, Liu et al., supra) were made using this method and tested as inhibitors of two cysteine proteases, papain and dipeptidyl amino-peptidase I (also called cathepsin C). However, the inhibition of papain by these .alpha.,.beta.-unsaturated compounds showed poor inhibition, evidenced by second order rate constants from less than 1 M.sup.-1 sec.sup.-1 to less than 70 M.sup.-1 sec.sup.-1 for the .alpha.,.beta.-unsaturated esters, and from less than 20 M.sup.-1 sec.sup.-1 to less than 60 M.sup.-1 sec.sup.-1 for the sulfone.
In addition, this chemistry has not been demonstrated with derivatives of .alpha.-amino acids other than those corresponding to glycine, or in the case of the ester, phenylalanine. Thus the chirality of these compounds is non-existent for the glycine derivatives and unclear for the phenylalanine derivatives. This is significant since inhibition of an enzyme generally requires a chiral compound.
Additional methods for selectively and irreversibly inhibiting cysteine proteases have relied upon alkylation by peptide .alpha.-fluoromethyl ketones (Rasnick, D., Anal. Biochem. (1985), 149, 416), diazomethylketones (Kirschke, H., Shaw, E. Biochem. Biphys. Res. Commun. (1981), 101, 454), acyloxymethyl ketones (Krantz, A. et al., Biochemistry, (1991), 30, 4678; Krantz, A. et al., U.S. Pat. No. 5,055,451, issued Oct. 8, 1991), and ketosulfonium salts (Walker, B., Shaw, E., Fed. Proc. Fed. Am. Soc. Exp. Biol., (1985), 44, 1433). The proposed mechanism of inactivation relies upon irreversible inactivation of the active site thiol group via alkylation, as depicted in Equation 2. ##STR2##
where
Other families of cysteine protease inhibitors include epoxysuccinyl peptides, including E-64 and its analogs (Hanada, K. et al., Agric. Biol. Chem (1978), 42, 523; Sumiya, S. et al., Chem. Pharm. Bull. ((1992), 40, 299 Gour-Salin, B. J. et al., J. Med. Chem., (1993), 36, 720), .alpha.-dicarbonyl compounds, reviewed by Mehdi, S., Bioorganic Chemistry, (1993), 21, 249, and N-peptidyl-O-acyl hydroxamates (Bromme, D., Neumann, U., Kirschke, H., Demuth, H-U., Biochim. Biophys. Acta, (1993), 1202, 271.